Cellular and Subcellular Distribution of Enzymes Involved in Aflatoxin Biosynthesis in Time-dependent Fractionated Colonies of Aspergillus parasiticus

Li-Wei Lee, Ching-Hsun Chiou, John Linz


Aflatoxins (AF) are highly toxic and carcinogenic secondary metabolites produced by the filamentous fungi Aspergillus flavus and A. parasiticus. AF Biosynthesis requires at least 18 enzymatic activities; the mechanisms for coordination of these activities are not clear. We hypothesize that AF enzymes are compartmentalized in the cytoplasm. The distribution of AF enzymes in colonies grown on solid medium was analyzed in A. parasiticus strain SU-1 (AF producing), AFS10 (aflR) and CS10 (wh-1, ver-1, pyrG). Colonies were divided into 3 fractions after 72 h of growth. Fraction S1, S2, and S3 contained cells grown for 72 h, 48 h, and 24 h, respectively. Protein extracts from each fraction were analyzed by Western blot using antibodies against AF enzymes Ver-1, OmtA and VBS. Fungal cells from each fraction were also fixed and embedded in paraffin or LR white resin. Thin sections were immunolabeled with fluorescence probes or gold conjugates for confocal laser scanning microscopy (CLSM) or transmission electron microscopy (TEM), respectively. Western blot analysis indicated that no AF proteins were detected in AFS10 which lacks an important transcription factor (AflR) required for AF synthesis. In SU-1, OmtA, VBS and Ver-1 were detected at highest levels in fraction S2. In fraction S3, the proteins appeared intact whereas proteins were degraded in fraction S1, indicating that proteolysis occurred as colony fraction aged. In contrast, VBS appeared intact in fraction S1. In CS10, most AF proteins were detected in fraction S3 and less in fractions S1 and S2. These data on protein abundance were consistent with results observed using immunofluorescence microscopy. Using CLSM, the fluorescence signals with all three probes appeared in patches within individual cells, indicating that the AF enzymes may be compartmentalized. The immunofluorescence pattern differed from the pattern in cells immunolabeled with anti-SKL (peroxisomal targeting signals) or in cells stained for nuclei. Preliminary TEM analysis localized OmtA to the cytoplasm; OmtA was not associated with specific membrane bound organelles. TEM analysis using anti-VBS and Ver-1 is underway.

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American Society for Microbiology General Meeting
    • ASM 102nd (2002)